CN108610402A - Applications of the peanut annexin Gene A hANN6 in improving plant and microorganism high temperature resistance and Oxidative Stress - Google Patents
Applications of the peanut annexin Gene A hANN6 in improving plant and microorganism high temperature resistance and Oxidative Stress Download PDFInfo
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- CN108610402A CN108610402A CN201810332804.XA CN201810332804A CN108610402A CN 108610402 A CN108610402 A CN 108610402A CN 201810332804 A CN201810332804 A CN 201810332804A CN 108610402 A CN108610402 A CN 108610402A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses peanut annexin genesAhANN6Application in improving plant and microorganism high temperature resistance and Oxidative Stress.The present invention passes through structureAhANN6The just over-express vector of gene, transformation mode plant Arabidopsis thaliana, the results showed that transgenic arabidopsis has stronger tolerance to high temperature stress, passes through structureAhANN6The prokaryotic expression carrier of gene converts Escherichia coli, the results showed that the gene can improve prokaryotes Escherichia coli high temperature resistant and oxidation resistant ability.The above results show peanut annexin geneAhANN6It is with a wide range of applications in terms of promoting plant and microorganism to resist high temperature and Oxidative Stress by genetic transformation and in terms of the agricultural productions such as crop anti-adversity is improved and microbiological industry.
Description
Technical field
The invention belongs to biotechnology, cultivate peanut annexin gene more particularly, to oneAhANN6In plant
And the application in microorganism high temperature resistance and Oxidative Stress.
Background technology
One of an important factor for high temperature is influence growth and development of plants and crop yield, plant is as fixation growth
Biology can not change the living environment of itself, however plant develops the defence machine of a series of complex in long-term evolutionary process
The extraneous changeable environment of system reply.Plant is to the reaction process and tolerance mechanism of high temperature usually with signaling molecule and with protection
The albumen of function is related, and heat-resisting relevant albumen can protect other albumen in plant to repair the albumen damaged from damage；It is anti-
Oxidase system Scavenger of ROS（reactive oxygen species, ROS）Ability, avoid cell and its membranous system by
To injury.
Peanut（Arachis hypogaea L.）It is worldwide Important Economic crop, is provided largely for the mankind
Edible oil and vegetable protein sources.The oil content of peanut seed is up to 40~60%, and protein content is 20~40%, carbon aquation
It is 10~20% to close object content.The nutritive value of peanut is high, and containing multivitamin and trace element, peanut also be used to eat
Product manufacturing industry and feed processing are international mostly important one of good economic trade crops.Peanut cultivation region is extensive,
There are cultivation in a countries and regions more than 100.Maximum cultivated area is Asia and Africa, and yield occupies global annual output
90%.The place of production of peanut includes the subtropical and tropical zones of hot climate, wherein many kinds to high temperature stress have it is very good
Adaptability
Annexin is widely distributed, miscellaneous a kind of soluble multifunctional protein family.1978, first animal membrane
Join albumen（Synexin, synexin）It is identified（Creutz C E, et al., Journal of Biological
Chemistry, 1978, 253:2858-2866）.1989, two calcium dependent lipid bindings are found that in tomato cell
Albumen, this is the annexin found for the first time in higher plant（Boustead C M, et al., FEBS Letters,
1989, 244:456-460）.Plant annexins are a Ca2+The cardiolipin binding protein of dependence, and can be given birth in plant
It plays a significant role in terms of long, development and environment stress（Zhou M L, et al., Functional & Integrative
Genomics, 2013, 13:241-251；Cantero A, et al., Plant Physiology &
Biochemistry, 2006, 44:13-24）.The experimental results show plant annexins wide participation to plant to non-
The response of biotic stress stress.
Currently, about peanut annexin geneAhANN6In plant and microorganism high temperature resistance and/or Oxidative Stress
Research have not been reported.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide peanut annexin genesAhANN6It is planted improving
Application in object and microorganism high temperature resistance and Oxidative Stress.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
The present invention is in peanut varieties Shanyou 523（Arachis hypogaeaL. Shanyou 523）Middle clone obtains peanut film
Join albumenAhANN6The global cDNA sequence of gene, nucleotide sequence such as SEQ ID NO：Shown in 1, the sequence is containing there are one big
Small is the open reading frame of 948 bp（ORF）.Open reading frame encodes the albumen of 315 amino acid residues, the ammonia of the albumen
Base acid sequence such as SEQ ID NO：Shown in 2.Sequence alignment analysis showsAhANN6Egg similar with soybean annexin in GenBank
White gene（NM_001255242）Homology be 84%；With clover annexin gene（XM_003598600）Homology be
83%；With cassava annexin gene（KM975564）Homology be 73%.
The present invention is also claimed for expandingAhANN6The primer pair of gene, including sense primer F and downstream primer R,
Its nucleotide sequence is successively such as SEQ ID NO:Shown in 3~4.
Forward primer：SEQ ID NO：3：
5’- TCCCCCGGGATGGCAACTCTTATTGCTCCCAGCA-3’
Reverse primer：SEQ ID NO：4：
5’- CGAGCTCTAGTCTTGTTTTCCCAACAATGTG-3’
By verifying peanut in transgenic arabidopsisAhANN6Gene function, resistance energy of the transgenic Arabidopsis plants to high temperature
Power enhances, and compared with the control group, the survival rate of transformed plant significantly improves after high temperature stress processing, and testing result is shown, with open country
Raw type plant is compared, and peroxidase in plant is converted（Peroxidase, POD, EC1.11.1.7）With ascorbic acid peroxide
Compound enzyme（Ascorbate peroxidase, APX, EC 1.111.11）On the expression quantity of two kinds of antioxidative defense enzymes is apparent
It rises.The above results showAhANN6Has the function of improving plant object to high temperature stress and oxidative stress resistivity.It willAhANN6Genetic transformation Escherichia coli is recombinantly expressed, and as a result shows recombination bacillus coli Rosette（DE3）To high temperature and right
Oxidant methyl amethyst（methyl viologen, MV）The resistivity of caused oxidative stress enhances, explanationAhANN6
Gene can also improve the ability of microorganism high temperature resistance and Oxidative Stress.
Therefore, SEQ ID NO：Gene shown in 1 and SEQ ID NO：The following application of albumen shown in 2 is protected in the present invention
In range.
SEQ ID NO：Peanut annexin gene shown in 1AhANN6Improving plant high temperature resistance and Oxidative Stress side
The application in face.
SEQ ID NO：Peanut annexin AhANN6 shown in 2 is in terms of improving plant high temperature resistance and Oxidative Stress
Using.
SEQ ID NO：Peanut annexin gene shown in 1AhANN6Improving microorganism high temperature resistance and Oxidative Stress
Application in terms of patience.
SEQ ID NO：Peanut annexin AhANN6 shown in 2 is improving microorganism high temperature resistance and Oxidative Stress patience
The application of aspect.
Compared with prior art, the invention has the advantages that：
（1）The present invention is separated to from peanutAhANN6Gene is proved by transformation mode plant Arabidopsis thalianaAhANN6It can be improved
Plant high temperature resistant coerces ability and improves the expression of antioxidase.The gene is applied to plant genetic engineering breeding, it is possible to
Improve resistivity of the important crops to high temperature and oxidative stress.
（2）The present invention is separated to from peanutAhANN6Gene is proved by converting prokaryotes Escherichia coliAhANN6
Resistivity of the microorganism to high temperature and oxidative stress can be improved.
（3）AhANN6 is and Ca2+The closely related memebrane protein of signal transduction, can be by responding high temperature and oxidative stress
Molecular signal conducts, and maintains normal membrane structure, is injured with reducing the environment stress suffered by cell, agriculture and micro-
There is important economic benefit and application prospect on biological industry.
Description of the drawings
Fig. 1 is peanutAhANN6 The amplification figure of cDNA sequence.M：DL2000 marker；1：AhANN6 Gene cDNA.
Fig. 2 is plant expression vector pRI101-AhANN6With coli expression carrier pET28a-AhANN6Structure signal
Figure.
Fig. 3 is in transgenic arabidopsisAhANN6The Q-PCR of gene is detected.WT represents wild type.OE-14, OE-15, OE-
16 respectively represent transgenosis overexpressionAhANN6Three strains.
Fig. 4 is that wild type and transgenosis are overexpressedAhANN6Arabidopsis high-temperature process phenotype.
Fig. 5 is that wild type and transgenosis are overexpressedAhANN6Arabidopsis survival rate after high-temperature process compares.
Fig. 6 is that wild type and transgenosis are overexpressedAhANN6Arabidopsis is after high-temperature processAtPODGene withAtAPXBase
The detection of expression of cause.
Fig. 7 is AhANN6 albumen in recombination bacillus coli Rosetta（DE3）The SDS-PAGE of middle expression detect and
Western-blot is detected.
Fig. 8 is conversionAhANN6Escherichia coli Rosetta（DE3）The treadmill test result of high temperature resistant and antioxidant assay
Fig. 9 is conversionAhANN6Escherichia coli Rosetta（DE3）In 46 DEG C and 75 μM L of high temperature-1Growth under MV processing is bent
Line.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
1 peanut of embodimentAhANN6The clone of gene cDNA sequence
（1）The preparation of peanut seed material：The peanut seed for harvesting the later stage of reaching maturity, strips cotyledon；
（2）Peanut seed Total RNAs extraction：Using Tiangeng biochemical technology（Beijing）The polysaccharide polyphenol plant total serum IgE of Co., Ltd carries
Kit is taken to extract；
（3）The synthesis of the first chains of cDNA：Using the PrimeScript of TaKaRa companiesTMRT reagent Kit with gDNA
Eraser（Perfect Real Time）Kit reverse transcription synthesizes the first chains of cDNA；
（4）AhANN6The clone of gene：Using peanut cDNA as template, cloned by PCRAhANN6CDNA segments, in primer
Increase restriction enzyme siteSmaI andSac I。
Forward primer：SEQ ID NO：3：
5’- TCCCCCGGGATGGCAACTCTTATTGCTCCCAGCA-3’
Reverse primer：SEQ ID NO：4：
5’- CGAGCTCTAGTCTTGTTTTCCCAACAATGTG-3’
PCR reaction systems：1 μ L cDNA templates, 1 μ L forward primers, 1 μ L reverse primers, 1 μ L dNTP（10 mM）, 5 μ L
10 × EX-Taq PCR Buffer, 0.5 ml EX-Taq enzymes（TaKaRa Products）, deionized water is finally supplemented, totality is made
Product is 50 ml.PCR programs：94 DEG C 3 minutes；Subsequently into following cycle：94 DEG C 25 seconds, 60 DEG C 30 seconds, 72 DEG C
1 minute, totally 10 recycled；Subsequently enter following cycle：94 DEG C 25 seconds, 52 DEG C 30 seconds, 72 DEG C 1 minute, totally 30
A cycle；Last 72 DEG C extend 7 minutes；
（5）The structure of plant expression vector：Amplification is completed to carry out agarose electrophoresis detection, recycles the band of suitable size.Sma I
WithSacI digestions use DNA QIAquick Gel Extraction Kits after the completion of digestion（QIAquick Gel Extraction Kit, QIAGEN companies
Product）It is recycled.Take 9.5 μ L recovery products and plant binary expression vector pRI101-AN（TaKaRa Products）It carries out
Connection, structure plant expression vector pRI101-AhANN6（Fig. 2）；
（6）Escherichia coli convert：Connection product converts bacillus coli DH 5 alpha competence（Tiangeng Products）, PCR, digestion to
Sequencing.Cell after conversion is containing kanamycins（50 mg/L）LB culture mediums on coated plate, be inverted in 37 DEG C and be incubated overnight,
Picking single bacterium colony is containing kanamycins（50 mg/L）LB liquid medium in cultivate, take a small amount of bacterium solution to carry out PCR identifications；
（7）The preparation of bacteria plasmid DNA：The thalline in above-mentioned LB liquid medium is collected, is carried using the plasmid of Tiangeng company is small
Kit prepares bacteria plasmid DNA.Sequencing is by Shanghai Invitrogen（Invitrogen）Company completes；
（8）The conversion of Agrobacterium tumefaciems：Correct pRI101- will be sequencedAhANN6Vector plasmid is transferred to crown gall agriculture by electric shocking method
Bacillus EHA105.
The genetic transformation of 2 arabidopsis of embodiment
One, transformation of Arabidopsis thaliana pre-treatment
Transformation of Arabidopsis thaliana pre-treatment, florescence arabidopsis, main tongue or side tongue are grown to 6-8 cm, remove and have fruit pod and bloomed
Flower bud, conversion the previous day are watered with water and keep high humidity environment.
Two, the preparation of Agrobacterium bacterium solution
（1）Agrobacterium EHA105 to 5 ml LB liquid medium of the inoculation with expression plasmid（Containing 50 mg/L of kanamycins and
30 mg/L of rifampin）Carry out the recovery and activation of strain, 200 rpm, 28 DEG C of shake cultures 24-36 hours；
（2）By 1：Bacterium solution is added to new LB liquid medium by 100 volume ratio（Containing kanamycins 50 mg/L and Li Fu
Flat 30 mg/L）In, 28 DEG C, 200 rpm shake cultures 16-20 hours, OD600 1.2-2.0；
（3）Room temperature collects thalline, and 5000 rpm centrifuge 10 minutes precipitation thalline；
（4）With the dip dyeing culture medium of Fresh（5 % sucrose solutions, addition 0.02-0.05 % surfactant Silwet L-
77）Thalline is resuspended to OD600 in 0.8-1.0.
Three, transformation of Arabidopsis thaliana
（1）Bacterium solution after resuspension is poured into clean beaker, the inflorescence part for the arabidopsis to be transformed handled well is immersed
45-60 seconds in beaker, residual dip dyeing liquid for shell is blotted after being placed 24 hours under dark condition with blotting paper and is put in normal culture
Under the conditions of continue to cultivate, dip dyeing is primary again as stated above after a week（Arabidopsis no longer converted before processing）, improve
Convert successful probability.
（2）Normal management plant collects ripe seed.Seed after harvest is containing 50 mg/L kanamycins and 150
Screening transgenic positive plant on the MS solid mediums of mg/L carbenicillins.
The Q-PCR of 3 genetically modified plants of embodiment is detected.
（1）The extraction of total serum IgE：Homozygous transgenic plant and wildtype Arabidopsis thaliana seed that screening obtains are seeded in 1/2
On MS culture mediums, cultivate after two weeks, according to Tiangeng biochemical technology（Beijing）The RNAprep Pure Plant Kit of Co., Ltd
（Polysaccharides&Polyphenolics-rich）Polysaccharide polyphenol plant total RNA extraction reagent box specification step carries
Take RNA；
（2）The synthesis of first chain cDNA：Using the PrimeScript of TaKaRa companiesTMRT reagent Kit with gDNA
Eraser（Perfect Real Time）, by specification step operation；
（3）Q-PCR reacts：
AhANN6Gene specific primer：
Forward primer：SEQ ID NO：5：5’- TTGCGGAAATCGCTTGTG -3’；
Reverse primer：SEQ ID NO：6：5’- AGCAGCCACATCTTCTTCCA -3’.
Reference geneActin2Primer：
Forward primer：SEQ ID NO：7：5’- CACTTGCACCAAGCAGCATGAAGA-3’；
Reverse primer：SEQ ID NO：8：5’- AATGGAACCACCGATCCAGACACT -3’.
The reaction system of quantitative fluorescent PCR is as follows：1 μ L, 2 × SYBR Green Mix of cDNA templates, 5 μ L, the examination
Agent is spun for Japan（Shanghai）Bio tech ltd's product, 0.5 μ L forward primers（10 μM of concentration）, 0.5 μ L reverse primers
（10 μM of concentration）, deionized water is finally supplemented, it is 10 μ L to make total volume.Using fluorescence quantitative PCR instrument（RoChe）
LightCycler480 carries out PCR reactions according to following response procedures：95 DEG C 1 minute；Subsequently into following cycle journey
Sequence, amplification 95 DEG C 10 seconds, 56 DEG C 10 seconds, 72 DEG C 20 seconds, totally 40 cycle.Quantitative PCR interpretation of result：Arabidopsis
The gene of middle stable expressionActin2 It is chosen as reference gene, sample data is carried out by the offline softwares of LightCycler480
Editor, using 2—ΔΔCTRelative quantitative analysis method carries out interpretation of result.The results are shown in Figure 3 by Q-PCR, does not have in wild-type plant
It detectsAhANN6Expression, and detected in 3 transgenic arabidopsis strains higherAhANN6Expression.
4 genetically modified plants of embodiment detect the tolerance of high-temperature process
1/2 MS culture mediums are seeded in after wild type and transgenic seed disinfection, after 4 DEG C of laminations 3 days, tablet is positioned over
Normal growing environment moved to Arabidopsis thaliana Seedlings in soil after 7 days（Vermiculite：Palm soil ratio 3：1）Growth 3 weeks.It will turnAhANN63 of gene are overexpressed strain and wildtype Arabidopsis thaliana seedling is positioned over 42 DEG C of illumination boxs, 16 small time
According to/8 hours dark cycles, 5000 Lx intensities of illumination were cultivated 60 hours, primary every watering in 12 hours therebetween.It has handled
Above-mentioned vegetable material is positioned over regular culture conditions culture 5 days by Cheng Hou, counts the survival rate after Stress treatment, and take pictures
Record.Test result shows that 3 transgenic line heat resistances compared with wild type significantly improve（Fig. 4）, extensive after high-temperature process
15 days multiple, the survival rate of genetically modified plants is apparently higher than wild-type plant（Fig. 5）.
5 plant anti-oxidation enzyme gene expression of embodiment detects
（1）The high-temperature process of plant：1/2 MS culture mediums are seeded in after wild type and transgenic seed disinfection, in 4 DEG C of laminations 3
After it, tablet is positioned over normal growing environment, after growing 5 days, after 45 DEG C of seedling high-temperature process 3 hours, is received immediately
It is cDNA to take sample, extraction RNA reverse transcriptions；
（2）Q-PCR is detected in arabidopsisAtPODGene andAtAPXThe expression of gene：
AtPODGene specific primer：（Wang M, et al., Plant Cell Reports, 2017, 36:1125-
1135）
Forward primer：SEQ ID NO：9：5’- CCAAACTCTTCGTGGACTATGC -3’；
Reverse primer：SEQ ID NO：10：5’- AACTCTTGGTCGCTCTGGAT -3’.
AtAPXGene specific primer：（Zhang L, et al., Acta Physiol Plant, 2014, 36:
1555-1564）
Forward primer：SEQ ID NO：11：5’- AATATGCTGCAGATGAGGATGC -3’；
Reverse primer：SEQ ID NO：12：5’- CAAGAATCAAGGAGGTAGGAGATG -3’.
Reference geneActin2Primer：
Forward primer：SEQ ID NO：7：5’- CACTTGCACCAAGCAGCATGAAGA -3’；
Reverse primer：SEQ ID NO：8：5’- AATGGAACCACCGATCCAGACACT -3’.
The reaction system of quantitative fluorescent PCR is as follows：1 μ L, 2 × SYBR Green Mix of cDNA templates, 5 μ L, the examination
Agent is spun for Japan（Shanghai）Bio tech ltd's product, 0.5 μ L forward primers（10 μM of concentration）, 0.5 μ L reverse primers
（10 μM of concentration）, deionized water is finally supplemented, it is 10 μ L to make total volume.Using fluorescence quantitative PCR instrument（RoChe）
LightCycler480 carries out PCR reactions according to following response procedures：95 DEG C 1 minute；Subsequently into following cyclic program,
Amplification 95 DEG C 10 seconds, 56 DEG C 10 seconds, 72 DEG C 20 seconds, totally 40 cycle.Quantitative PCR interpretation of result：It is steady in arabidopsis
Surely the gene expressedActin2 It is chosen as reference gene, sample data editor is carried out by the offline softwares of LightCycler480,
Using 2—ΔΔCTRelative quantitative analysis method carries out interpretation of result.
Measurement result is shown, compared with wild type, in transgenic arabidopsisAtAPXWithAtPODIt is expressed after high-temperature process
Amount up-regulation（Fig. 6）, explanationAhANN6In the controllable arabidopsis of gene with the relevant protection enzyme gene expression of anti-oxidant approach,AhANN6The antioxidant capacity of plant can be also improved while improving tolerance of the quasi- plant to high temperature.
Embodiment 6AhANN6Recombinant expression in Escherichia coli and detection
（1）The structure of prokaryotic expression carrier：
With pRI101-AhANN6Vector plasmid is template, increases restriction enzyme site in primerBamHI andXhoI is cloned by PCR
It obtainsAhANN6CDNA segments.
Forward primer：SEQ ID NO：13：
5’- CGCGGATCCGCGATGGCAACTCTTATTGCTCCC -3’
Reverse primer：SEQ ID NO：14：
5’- CCGCTCGAGCGGCTAGTCTTGTTTTCCCAACAATGTG -3’
PCR reaction systems：1 μL pRI101-AhANN6Plasmid template, 1 μ L forward primers（10 μM of concentration）, 1 μ L reversely draw
Object（10 μM of concentration）, 1 μ L dNTP (10 mM), 5 μ L 10 × EX-Taq PCR Buffer, 0.5 ml EX-Taq enzymes
（TaKaRa Products）, deionized water is finally supplemented, it is 50 ml to make total volume.PCR programs：94 DEG C 3 minutes；Then into
Enter following cycle：94 DEG C 25 seconds, 60 DEG C 30 seconds, 72 DEG C 1 minute, totally 10 cycle；Entering following cycle：94
DEG C 25 seconds, 52 DEG C 30 seconds, 72 DEG C 1 minute, totally 30 cycles；Last 72 DEG C extend 7 minutes.
Amplification is completed to carry out agarose electrophoresis detection, recycles the band of suitable size.BamH I、XhoI digestions, digestion are complete
Cheng Houyong DNA QIAquick Gel Extraction Kits（QIAquick Gel Extraction Kit, QIAGEN Products）It is recycled.Take 9.5
μ L recovery products are attached with prokaryotic expression carrier pET28a, build pET28a-AhANN6Carrier（Fig. 2）；
（2）Escherichia coli convert：Connection product converts bacillus coli DH 5 alpha competence（Tiangeng biochemical technology（Beijing）Co., Ltd
Tiangeng Products）, PCR, digestion is being sequenced.Cell after conversion is containing kanamycins（50 mg/L）And chloramphenicol（34
mg/L）LB culture mediums on coated plate, be inverted in 37 oC and be incubated overnight, picking single bacterium colony is containing kanamycins（50 mg/L）With
Chloramphenicol（34 mg/L）LB liquid medium in cultivate, take a small amount of bacterium solution to carry out PCR identifications, be sequenced by Shanghai Invitrogen
（Invitrogen）Company completes；
Embodiment 7 convertsAhANN6SDS-PAGE the and Western Blot detections of recombination bacillus coli
One, e. coli protein induced expression
（1）The recovery of Escherichia coli：Control plasmid pET28a and plasmid pET28a- will be containedAhANN6Convert Escherichia coli
Rosetta（DE3）, 37 DEG C/150 rpm take 30 μ L coatings to contain kanamycins after recovering 1 hour（50 mg/L）It is mould with chlorine
Element（34 mg/L）LB tablets；
（2）A small amount of cultures of Escherichia coli：In monoclonal colonies to 5 ml LB liquid mediums on picking tablet, 37 DEG C,
180 rpm shake bacterium and stay overnight；
（3）The mass propgation of Escherichia coli：Bacterium will be stayed overnight according to 1：100 ratio is inoculated into containing 10 ml LB liquid mediums
Conical flask in, 37 DEG C shake bacterium to OD600 be 0.6；
（4）Protein expression induces：Bacterium solution reaches OD600 when being 0.6, is added the IPTG of final concentration of 0.1 mM into bacterium solution, and 37
Bacterium culture is shaken at DEG C, takes 1 ml bacterium solutions for SDS-PAGE every 2 hours（It takes to 8 hours and terminates）.
Two, prokaryotic expression bacterial protein extracts
（1）By the front and back bacterium solution of induction, 13200 rpm are centrifuged 1 minute at room temperature, and incline supernatant, and 50 are then added thereto
μL PBS（1.06 mM KH2PO4、155.17 mM NaCl、2.97 mM Na2HPO4, pH value 7.4）, thalline is fully suspended
15 μ L 5 × albumen sample-loading buffers are added after getting up（6.8,1 M Tris-HCl of pH 6.25 ml, SDS 2.5 g, β-
6.25 ml of mercaptoethanol, 12.5 ml of glycerine, 10 mg of bromophenol blue, 25 ml are settled to, in 4 DEG C of preservations）；
（2）After sample vortex oscillation is mixed well, 100 DEG C of boiling water boilings 5 minutes, under room temperature, 13200 rpm centrifugations 20
Minute, take supernatant；
（3）By the protein example of extraction（The front and back bacterial protein of induction）It is detached by SDS-PAGE, coomassie brilliant blue staining
Detect target protein.
Three, SDS-PAGE electrophoresis and Western blot
SDS-PAGE electrophoresis is with Western blot with reference to the method for Serrano-Maldonado etc.（Serrano-Maldonado
et al., Journal of Molecular Microbiology and Biotechnology, 2018, 28:14-27）.
Used primary antibody is the mouse anti-His of Wuhan Sanying Bio-Technology Co., Ltd. in Western blot
Monoclonal antibody, secondary antibody Ai Bokang（Shanghai）The Goat pAb to Ms IgG of trade Co., Ltd（AP）.
NBT/BCIP（Perkin-Elmer, Waltham, MA, USA）As developer to display target band.
SDS-PAGE is the results show that pET28a zero loads bacterium is expressed without destination protein before and after IPTG inductions are added；
PET28a- before IPTG inductionsAhANN6There is no destination protein expression in recombinant bacterium, destination protein great expression after induction, until the 4th
Hour inducing amount reaches maximum, and inducible protein matter band is located at 35 KDa and the theoretical molecular weight 36.2 of AhANN6 albumen
KDa is consistent, and Western blot results, which are shown at 36 KDa, detects the extremely strong protein signal of signal, illustrates that peanut film joins
Albumin A hANN6 is in expression in escherichia coli success.
Embodiment 8 convertsAhANN6Recombination bacillus coli high temperature resistance and oxidative stress analysis
One, pET28a-AhANN6Convert the tablet experiment of recombinant bacterium
（1）A small amount of cultures of Escherichia coli：Picking recombinant bacterium and control strain contain kanamycins to 5 ml（50 mg/L）With
Chloramphenicol（Dense 34 mg/L）LB culture mediums in, 180 rpm, 37 DEG C of shaken cultivations are stayed overnight；
（2）The mass propgation of Escherichia coli：According to 1：100 ratio is inoculated into the 50 ml tapers equipped with 10 ml LB culture mediums
In bottle, culture medium contains kanamycins（50 mg/L）And chloramphenicol（34 mg/L）, 37 DEG C, 180 rpm shaken cultivations about 3 are small
Reach 0.5 up to OD600.
（3）Protein expression induces：IPTG to final concentration of 0.1 mmol/L is added, 37 DEG C of shaken cultivations 3 are then continued to
OD600 is diluted to 1.0 by h, and on this basis according to 10-1、10-2、10-3、10-4、10-5Concentration gradient be diluted.
（4）Identify tolerance of the recombinant bacterium to high temperature：Each sample respectively takes 5 μ L to be added drop-wise on LB solid plates, culture temperature
Degree is 42 DEG C.
（5）Identify resistance of the recombinant bacterium to oxidative stress：Each sample respectively takes 5 μ L to be added drop-wise to the LB of the MV containing 75 μM/L
On solid plate, cultivation temperature is 37 DEG C.
Two, pET28a-AhANN6Convert the measurement of recombinant bacterium growth curve
（1）A small amount of cultures of Escherichia coli：Picking recombinant bacterium and control strain contain kanamycins to 5 ml（50 mg/L）With
Chloramphenicol（34 mg/L）LB culture mediums in, 180 rpm, 37 DEG C of shaken cultivations are stayed overnight；
（2）The mass propgation of Escherichia coli：According to 1：100 ratio is inoculated into the 50 ml tapers equipped with 10 ml LB culture mediums
In bottle, culture medium contains kanamycins（50 mg/L）And chloramphenicol（34 mg/L）, 37 DEG C, 180 rpm shaken cultivations about 3 are small
Reach 0.5 up to OD600；
（3）Protein expression induces：IPTG to final concentration of 0.1 mmol/L is added, 37 DEG C of 3 h of shaken cultivation are then continued to, it will
OD600 is diluted to 0.8；
（4）Detect influence of the oxidative stress to recombinant bacterium：According to 1：100 ratio is inoculated into 20 ml containing 75 μM/L MV
In LB culture mediums, culture medium contains kanamycins（50 mg/L）And chloramphenicol（34 mg/L）, 37 DEG C, 180 rpm shaken cultivations
12 hours, during which bacterium solution OD600 values were measured every 2 hours.
（5）Influence of the high temperature to recombinant bacterium is detected, according to 1：Bacterium solution is inoculated into 20 ml LB Liquid Cultures by 100 ratio
In base, 46 DEG C, during which 180 rpm shaken cultivations 12 hours measured bacterium solution OD600 values every 2 hours.Flat-plate experimental result
（Fig. 8）The result measured with growth curve（Fig. 9）Show to express peanut annexin geneAhANN6Recombinant bacterium compare
There are stronger high temperature resistant and oxidation resistance according to bacterium.
Sequence table
<110>Zhongshan University
<120>Applications of the peanut annexin Gene A hANN6 in improving plant and microorganism high temperature resistance and Oxidative Stress
<130> YG18102299AA042
<141> 2018-04-13
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 948
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 1
atggcaactc ttattgctcc cagcaaccat tcctcagctg aagatgctga agctctccaa 60
aaagcattca aaggatgggg agctgatgat aagaccatta tagcaattct tggacacaga 120
aatgttcatc agaggcagca aatcagaaaa gcttatgagg agcttcacca agaggatctc 180
attaagcgac tcgaatccga gatctctggt gactttgaga gagctatgta tcgttggatg 240
ttggaacctg ctgatcgaga tgctgttcta gcgaatgtag caatcagaaa tgggaagaaa 300
gattttcatg taattgcgga aatcgcttgt gttttatctg ctgaagagct tttggcagtg 360
aggcgtgcct atcgccaccg ctacaagcgt tccttggaag aagatgtggc tgctaacacc 420
actggccacc ttcgcgagct tttggttgga ttagtaagct catttaggta tgagggtgat 480
gagataaatg caagacttgc acagagtgaa gccaatattc tccatgaaac tgtgaaagag 540
aagaaaggaa actatgaaga agccattagg attcttacta caagaagcaa gactcagctt 600
gttgctactt tcaaccgcta cagagatgag catgccattt ccatcagcaa gaaattgcta 660
gacaatcaag cttctgatga tttctacaag gcattgcaca ctgcaattcg ttgcatcaat 720
gaccacaaaa agtactatga aaaggttctg cgcaacgcga taaaaaaggt tgggaccgac 780
gaggatgcgc tgagccgagt cgtggtgaca agggctgaga aggatcttag ggacatcaaa 840
gaactgtatt acaagagaaa cagtgttcat cttgaggatg ctgttgccaa agaaacttca 900
ggggactaca agaagttcct cctcacattg ttgggaaaac aagactag 948
<210> 2
<211> 315
<212> PRT
<213>Peanut (Arachis hypogaea L.)
<400> 2
Met Ala Thr Leu Ile Ala Pro Ser Asn His Ser Ser Ala Glu Asp Ala
1 5 10 15
Glu Ala Leu Gln Lys Ala Phe Lys Gly Trp Gly Ala Asp Asp Lys Thr
20 25 30
Ile Ile Ala Ile Leu Gly His Arg Asn Val His Gln Arg Gln Gln Ile
35 40 45
Arg Lys Ala Tyr Glu Glu Leu His Gln Glu Asp Leu Ile Lys Arg Leu
50 55 60
Glu Ser Glu Ile Ser Gly Asp Phe Glu Arg Ala Met Tyr Arg Trp Met
65 70 75 80
Leu Glu Pro Ala Asp Arg Asp Ala Val Leu Ala Asn Val Ala Ile Arg
85 90 95
Asn Gly Lys Lys Asp Phe His Val Ile Ala Glu Ile Ala Cys Val Leu
100 105 110
Ser Ala Glu Glu Leu Leu Ala Val Arg Arg Ala Tyr Arg His Arg Tyr
115 120 125
Lys Arg Ser Leu Glu Glu Asp Val Ala Ala Asn Thr Thr Gly His Leu
130 135 140
Arg Glu Leu Leu Val Gly Leu Val Ser Ser Phe Arg Tyr Glu Gly Asp
145 150 155 160
Glu Ile Asn Ala Arg Leu Ala Gln Ser Glu Ala Asn Ile Leu His Glu
165 170 175
Thr Val Lys Glu Lys Lys Gly Asn Tyr Glu Glu Ala Ile Arg Ile Leu
180 185 190
Thr Thr Arg Ser Lys Thr Gln Leu Val Ala Thr Phe Asn Arg Tyr Arg
195 200 205
Asp Glu His Ala Ile Ser Ile Ser Lys Lys Leu Leu Asp Asn Gln Ala
210 215 220
Ser Asp Asp Phe Tyr Lys Ala Leu His Thr Ala Ile Arg Cys Ile Asn
225 230 235 240
Asp His Lys Lys Tyr Tyr Glu Lys Val Leu Arg Asn Ala Ile Lys Lys
245 250 255
Val Gly Thr Asp Glu Asp Ala Leu Ser Arg Val Val Val Thr Arg Ala
260 265 270
Glu Lys Asp Leu Arg Asp Ile Lys Glu Leu Tyr Tyr Lys Arg Asn Ser
275 280 285
Val His Leu Glu Asp Ala Val Ala Lys Glu Thr Ser Gly Asp Tyr Lys
290 295 300
Lys Phe Leu Leu Thr Leu Leu Gly Lys Gln Asp
305 310 315
<210> 3
<211> 34
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 3
tcccccggga tggcaactct tattgctccc agca 34
<210> 4
<211> 31
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 4
cgagctctag tcttgttttc ccaacaatgt g 31
<210> 5
<211> 18
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 5
ttgcggaaat cgcttgtg 18
<210> 6
<211> 20
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 6
agcagccaca tcttcttcca 20
<210> 7
<211> 24
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 7
cacttgcacc aagcagcatg aaga 24
<210> 8
<211> 24
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 8
aatggaacca ccgatccaga cact 24
<210> 9
<211> 22
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 9
ccaaactctt cgtggactat gc 22
<210> 10
<211> 20
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 10
aactcttggt cgctctggat 20
<210> 11
<211> 22
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 11
aatatgctgc agatgaggat gc 22
<210> 12
<211> 24
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 12
caagaatcaa ggaggtagga gatg 24
<210> 13
<211> 33
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 13
cgcggatccg cgatggcaac tcttattgct ccc 33
<210> 14
<211> 37
<212> DNA
<213>Peanut (Arachis hypogaea L.)
<400> 14
ccgctcgagc ggctagtctt gttttcccaa caatgtg 37
Claims (6)
1.SEQ ID NO：The gene of peanut annexin shown in 1AhANN6Improving plant and/or microorganism high temperature resistance and antioxygen
Change the application in stress.
2.SEQ ID NO：The AhANN6 of peanut annexin shown in 2 is improving plant and/or microorganism high temperature resistance and and/or antioxygen
Change the application in stress.
3. application according to claim 1, which is characterized in that the application is structure peanut annexinAhANN6Gene
Just over-express vector, be transformed into plant；Or structure peanut annexinAhANN6The prokaryotic expression carrier of gene, conversion
Into microorganism.
4. application according to claim 3, which is characterized in that the justice over-express vector is pRI101-AhANN6, former
Nuclear expression carrier is pET28a-AhANN6。
5. application according to claim 3, which is characterized in that the plant is arabidopsis.
6. application according to claim 3, which is characterized in that the microorganism is Escherichia coli.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810332804.XA CN108610402B (en) | 2018-04-13 | 2018-04-13 | Application of peanut annexin gene AhANN6 in improving high temperature resistance and oxidation stress resistance of plants and microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810332804.XA CN108610402B (en) | 2018-04-13 | 2018-04-13 | Application of peanut annexin gene AhANN6 in improving high temperature resistance and oxidation stress resistance of plants and microorganisms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108610402A true CN108610402A (en) | 2018-10-02 |
| CN108610402B CN108610402B (en) | 2021-05-25 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810332804.XA Active CN108610402B (en) | 2018-04-13 | 2018-04-13 | Application of peanut annexin gene AhANN6 in improving high temperature resistance and oxidation stress resistance of plants and microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108610402B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444161A (en) * | 2021-06-29 | 2021-09-28 | 南京农业大学 | Radish annexin RsANN1a and coding gene and application thereof |
| CN116555322A (en) * | 2023-01-17 | 2023-08-08 | 中国科学院华南植物园 | TtanxNL gene and application of coded protein thereof |
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| TW201008505A (en) * | 2008-08-20 | 2010-03-01 | Nat Univ Chiayi | Bioactive peanut sprout powders bearing lifespan extension and anti-oxidative activities, its preparation, composition and products containing the active compounds |
| CN102229662A (en) * | 2011-06-14 | 2011-11-02 | 中山大学 | Lotus annexin and expression vector and application thereof |
| CN102482682A (en) * | 2009-08-25 | 2012-05-30 | 巴斯夫植物科学有限公司 | Nematode-resistant transgenic plants |
| CN102770542A (en) * | 2007-05-22 | 2012-11-07 | 巴斯夫植物科学有限公司 | Plants with increased tolerance and/or resistance to environmental stress and increased biomass production |
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| CN102770542A (en) * | 2007-05-22 | 2012-11-07 | 巴斯夫植物科学有限公司 | Plants with increased tolerance and/or resistance to environmental stress and increased biomass production |
| TW201008505A (en) * | 2008-08-20 | 2010-03-01 | Nat Univ Chiayi | Bioactive peanut sprout powders bearing lifespan extension and anti-oxidative activities, its preparation, composition and products containing the active compounds |
| CN102482682A (en) * | 2009-08-25 | 2012-05-30 | 巴斯夫植物科学有限公司 | Nematode-resistant transgenic plants |
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| MEIJING HE,等: "Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.)", 《GENE》 * |
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| 佚名: "annexin-like protein RJ4 isoform X1 [Arachis ipaensis], XP_016179945.1", 《GENBANK》 * |
| 黎茵，等: "种子热稳定蛋白的研究进展", 《北方园艺》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444161A (en) * | 2021-06-29 | 2021-09-28 | 南京农业大学 | Radish annexin RsANN1a and coding gene and application thereof |
| CN116555322A (en) * | 2023-01-17 | 2023-08-08 | 中国科学院华南植物园 | TtanxNL gene and application of coded protein thereof |
| CN116555322B (en) * | 2023-01-17 | 2023-10-27 | 中国科学院华南植物园 | TtanxNL gene and application of coded protein thereof |
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| Publication number | Publication date |
|---|---|
| CN108610402B (en) | 2021-05-25 |
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